Esophageal cancer is the 6th most common malignant neoplasm worldwide with more than 90% of all cases being esophageal squamous cell carcinoma (SCC). Risk factors for esophageal SCC include tobacco use, alcohol consumption, nutritional deficiency, and intake of food contaminated with various mycotoxins. Esophageal SCC grows more quickly than other kinds of gastrointestinal malignancies and patients with this disease have a very poor prognosis. Although surgery, chemotherapy, and radiotherapy alone or combined are used, the overall 5-year survival rate for this disease is still very low, ranging from 5% to 15%. To decrease the incidence of esophageal cancer, cancer chemoprevention through dietary and/or chemical intervention would be a logical and practical approach. Numerous compounds have been tested; however, the trails to date have not resulted in a decrease of esophageal cancer incidence. There is an urgent need to identify protective agents, which can prevent this disease in the individuals associated with increasing esophageal cancer risk. The long-term goal of our studies is to develop mechanism-driven safe and effective prevention strategies for reducing the incidence of esophageal cancer in high-risk populations. N-nitrosomethylbenzylamine (NMBA)- induced rat preclinical model of esophageal cancer has been used extensively to investigate the mechanisms of esophageal carcinogenesis and to evaluate the efficacy of potential chemopreventive agents. The multistage evolution of esophageal SCC, from normal to hyperplasia, dysplasia and papilloma, is ideal for the application of chemoprevention strategies. In this proposal, we outline our strategy to develop combinational approaches to the chemoprevention of esophageal cancer. Our central hypothesis is that combination of cyclooxygenase-2 (COX-2) inhibitor, celecoxib, inducible nitric oxide synthase (iNOS) inhibitor, S,S'-1,4- phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT), and freeze-dried black raspberries (BRB) will increase efficacy against tumor development while minimizing toxicity in NMBA-rat preclinical model. The specific aims are: 1. To establish strategies to improve efficacy of esophageal cancer prevention by a combination of COX-2 and iNOS inhibitors; 2. To establish strategies to improve efficacy of esophageal cancer prevention by a combination of pure compound(s) and natural food products and to determine the cellular and molecular mechanisms of their actions; 3. To identify cellular and molecular biomarkers of NMBA-induced tumorigenesis in the rat esophagus. Overall, a successful outcome of this hypothesis-driven preclinical study will provide important information and rationale to develop a combination with agents that may have synergistic activity in human clinical trials of chemoprevention of esophageal cancer. Project Narrative The overall objective of this project is to evaluate the efficacy of combinational approaches against esophageal cancer development, to investigate the mechanistic basis of these agents, and to identify cellular and molecular biomarkers of esophageal tumorigenesis using a rat preclinical model, which is highly analogous to human esophageal squamous cell carcinoma.